Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA.

Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.     

薄鳞鱼类化石的新发现及其地层意义

本文记述了薄鳞鱼类(leptolepids)一新属新种——罗家峡隆德鱼(Longdeichthys luojiaxiaensis gen.et sp.nov.)。它和广泛分布于我国北方的另一原始真骨鱼类狼鳍鱼(Lycoptera)共生。因而,为研究真骨鱼类的演化和确定我国北方中生代含鱼岩系的时代及地层对比上,提供了新的资料。     

On the Nature of the Interaction Between Serotonin and Serotonin Binding Protein: Effect of Nucleotides, Ions, and Sulfhydryl Reagents

Abstract: Rat brain serotonin binding protein (SBP) was found to have essential -S-S and -SH groups. Both reduction of the disulfide bond by dithiothreitol or mercaptoethanol and modification of -SH group(s) by Ellman reagent or alkylating agents caused loss of binding capacity. In contrast, formation of a mixed disulfide bond with sodium metabisulfite did not affect the binding capacity. Serotonin in the presence of Fe²⁺ and phosphate was found to bind to either an -SH group or to a site in very close proximity. Addition of serotonin protected -SH groups from modification by Ellman reagent and from denaturation of protein upon storage. Lipids that enhance binding of serotonin to SBP also protected -SH groups from modification. Nucleotides were found to be strong inhibitors of the binding of serotonin to SBP. The inhibitory effect of nucleotides was due to their chelating properties and not to their ability to phosphorylate the protein or to bind directly to it. Inhibition by nucleotides and other chelators was reversible. Binding capacity was fully restored after removal of the chelator by molecular sieve chromatography and addition of Fe²⁺. The ionic environment had a marked effect on the binding: intracellular ions such as K⁺ were found to enhance the binding, and extracellular ions such as Na⁺ and Ca²⁺ inhibited the binding. Based on these data and our previous studies, we suggest that SBP is an intracellular protein that acts as a storage protein. Consistent with our data is formation of a complex of SBP-S-Fe-S that in a hydrophobic surrounding could bind up to four molecules of serotonin in coordination bond with Fe²⁺ and thereby reduce the osmotic pressure within a storage vesicle. Extracellular ionic conditions that favor the dissociation of the complex would free the amine to interact with its receptor or the presynaptic reuptake carrier.     

Comparative bioenergetics of sulfate reduction in Desulfovibrio and Desulfotomaculum spp.

Extracts of Desulfotomaculum nigrificans, Desulfotomaculum orientis, and Desulfotomaculum ruminis exhibit low levels of inorganic pyrophosphatase but were found to have high levels of pyrophosphate:acetate phosphotransferase. Conversely, extracts of Desulfovibrio gigas, Desulfovibrio vulgaris, and Desulfovibrio desulfuricans Norway 4 were shown to have high levels of inorganic pyrophosphatase but negligible amounts of pyrophosphate:acetate phosphotransferase. Both enzymes are reductant activated and appear to have an analogous function in removing pyrophosphate formed during the activation of sulfate. Conservation of the bond energy of pyrophosphate in Desulfotomaculum eliminates the necessity for invoking electron-transfer-coupled phosphorylation to account for the growth of these bacteria on lactate plus sulfate. Relative growth yields of Desulfovibrio vulgaris and Desulfotomaculum orientis on lactate plus sulfate indicate that the latter does not carry out significant electron-transfer-coupled phosphorylation in this mode of growth.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Ca2+-stimulated ribonuclease. A new marker enzyme of differentiated rat mammary tissues.

A unique ribonuclease, active only in the presence of Ca2+, was present in lactating mammary gland and milk of the rat. This enzyme was absent from virgin-rat mammary gland and non-mammary tissues of lactating rats. The presence of moderate activity in differentiated mammary tumours, together with an increase in activity in normal tissue parelleling development of mammary function, identify this enzyme as a marker of mammary differentiation in the rat.